Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo-and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo-and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family. H eptahelical receptors (HHRs) constitute the largest single family of transmembrane signaling molecules that respond to diverse external stimuli such as hormones, neurotransmitters, chemoattractants, odorants, and photons. Although these receptors have been generally thought to function as monomeric entities, there is growing evidence that a number of HHRs assemble as functional homo-and heterooligomers (1, 2). Dimerization seems to be necessary for function of the class C subfamily of HHRs comprising the metabotropic glutamate, calcium sensing, the GABAB, and pheromone receptors that are targeted to the plasma membrane as preformed dimers which are stabilized by ligand binding (3-7). Several HHRs such as somatostatin receptors (SSTRs), dopamine receptors, gonadotrophin-releasing hormone receptor (GnRHR), luteinizing hormone͞chorionic gonadotrophin hormone receptor, and chemokine receptors, however, which belong to the rhodopsin-like class A subfamily of HHRs, assemble on the membrane as homo-and heterooligomeric species in response to agonist activation (8-15).In the case of SSTRs, we have shown by photobleaching fluorescence resonance energy transfer (pbFRET) that the human (h) type 5 receptor (hSSTR5 or R5) exists in the basal state as a monomer, and that activation by ligand induces dose-dependent oligomerization (8). When coexpressed with another SSTR (hSSTR1 or R1) or an unrelated HHR such as the dopamine 2 receptor (D 2 R), R5 also forms a heterooligomer that displays pharmacological properties distinct from those of either of the separate receptors (9). Little is known about the stoichiometry of ligand-receptor reactions or the specificity for homoand heterooligomeric interactions between two receptors that are coexpressed in the same cell. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with R5 or R1, or cotransfected with R5 and R1, we have analyzed the ...

Calcium-sensing receptors (CaSRs) regulate systemic calcium homeostasis in the parathyroid gland, kidney, intestine, and bone and translate fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. The CaSR is a G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor that operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. Here, we used multiwavelength total internal reflection fluorescence microscopy to demonstrate that the signaling properties of the CaSR result from agonist-driven maturation and insertion of CaSRs into the plasma membrane. Plasma membrane CaSRs underwent constitutive endocytosis without substantial recycling, indicating that signaling was determined by the rate of insertion of CaSRs into the plasma membrane. Intracellular CaSRs colocalized with calnexin in the perinuclear endoplasmic reticulum and formed complexes with 14-3-3 proteins. Ongoing CaSR signaling resulted from agonist-driven trafficking of CaSR through the secretory pathway. The intracellular reservoir of CaSRs that were mobilized by agonist was depleted when glycosylation of newly synthesized receptors was blocked, suggesting that receptor biosynthesis was coupled to signaling. The continuous, signaling-dependent insertion of CaSRs into the plasma membrane ensured a rapid response to alterations in the concentrations of extracellular calcium or allosteric agonist despite ongoing desensitization and endocytosis. Regulation of CaSR plasma membrane abundance represents a previously unknown mechanism of regulation that may be relevant to other receptors that operate in the chronic presence of agonist.